Acetylation of PKA modulates both its catalytic activity and anchoring to scaffolding AKAP4 protein.

GENTILE, IÑAKI; NOVERO, ANALIA G.; CURCIO, CATALINA; BUFFONE, MARIANO G.; BINOLFI, ANDRES; KRAPF, DARIO; STIVAL, CINTIA

Congreso; LIX Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2023

After ejaculation, mammalian sperm require a period of residence in the female reproductive tract to gain fertilizing competence, in a process named “capacitation”. Capacitation can also be achieved in vitro by incubating sperm in a defined culture medium that induces an increase in cAMP levels which directly activates Protein Kinase A (PKA). Current knowledge positions PKA as a key player of sperm capacitation. In vivo, the resulting PKA activity depends on its catalytic activity, proper positioning, and activity of ser/thr phosphatases. Understanding its fine-tuned regulation becomes even more relevant after we recently demonstrated how cAMP levels decrease after 10 minutes of incubation in a capacitating medium, while enzymatic levels of PKA remain constant. Two studies have identified 576 and 456 acetylated proteins in capacitated and non-capacitated human spermatozoa respectively, revealing a pool of proteins that are acetylated either only in capacitating or in non-capacitating conditions. Among these proteins, the catalytic subunit of PKA (cPKA) was identified to be acetylated in human capacitated sperm. We have confirmed its acetylation in murine sperm by western-blot experiments. Moreover, a recombinant his-tagged version of the mouse cPKA (“cPKArec”) was purified in order to analyze the effect of lysine acetylation in this kinase. The recombinant cPKA was acetylated using whole sperm extracts supplemented with Acetyl CoA and inhibitors of deacetylases. Our results showed that acetylated cPKArec increased its catalytic activity, as measured in vitro using the peptidic substrate Kemptide and a gel mobility shift assay. In addition, the acetylating extract not only induced acetylation of cPKA but also stimulated phosphorylation of cPKArec in Thr197, which has been associated with PKA activation. We then aimed at understanding structural modifications promoted by Lys acetylation of cPKA in the recombinant protein. We conducted NMR experiments on a 600 MHz Nuclear Magnetic Resonance Spectrometer and observed that acetylation induced spectral shifts in amino acids of the catalytic site, which might account for its augmented activity.Acetylation of cPKArec also affected anchoring since affinity purification of His-tagged cPKA showed an increased interaction of AKAP4 with acetylated cPKArec. This interaction protected AKAP4 from degradation during capacitation, when mouse sperm cells were incubated with deacetylase inhibitors, as shown by western-blot experiments. Our results shed light onto the molecular modulation of PKA promoted by Lys acetylation that are independent of cAMP, and might have key roles, not only in sperm, but in general cell physiology.